Pilot study of humanized glypican-3-targeted zirconium-89 immuno-positron emission tomography for hepatocellular carcinoma

Purpose: Glypican-3 (GPC3)-targeted radioisotope immuno-positron emission tomography (immunoPET) may lead to earlier and more accurate diagnosis of hepatocellular carcinoma (HCC), thus facilitating curative treatment, decreasing early recurrence, and enhancing patient survival. We previously demonstrated reliable HCC detection using a zirconium-89-labeled murine anti-GPC3 antibody (89Zr-αGPC3M) for immunoPET. This study evaluated the efficacy of the humanized antibody successor (αGPC3H) to further clinical translation of a GPC3-based theranostic for HCC. Methods: In vitro αGPC3 binding to HepG2 cells was assessed by flow cytometry. In vivo 89Zr-αGPC3H and 89Zr-αGPC3M tumor uptake was evaluated by PET/CT and biodistribution studies in an orthotopic xenograft mouse model of HCC. Results: αGPC3H maintained binding to GPC3 in vitro and 89Zr-αGPC3H immunoPET identified liver tumors in vivo. PET/CT and biodistribution analyses demonstrated high 89Zr-αGPC3H tumor uptake and tumor-to-liver ratios, with no difference between groups. Conclusion: Humanized αGPC3 successfully targeted GPC3 in vitro and in vivo. 89Zr-αGPC3H immunoPET had comparable tumor detection to 89Zr-αGPC3M, with highly specific tumor uptake, making it a promising strategy to improve HCC detection.


Introduction
Hepatocellular carcinoma (HCC) is increasing in incidence worldwide and has become the fastest growing cause of cancer death in the United States, with a median survival of less than one year [1][2][3].In order to improve survival with current treatments, HCC must be detected early when it is amenable to surgical resection or transplantation [3.4].However, multiphase, computed tomography (CT) or magnetic resonance imaging frequently misses lesions less than 1 cm, resulting in diagnostic uncertainty, delayed diagnosis, and early recurrence following resection [5,7].Innovative technology capable of detecting HCC with enhanced sensitivity and speci city is therefore imperative and pressing.
Our group previously demonstrated that immunoPET using zirconium-89 ( 89 Zr)-labeled murine antibody targeting GPC3 ( 89 Zr-αGPC3 M ) reliably identi ed small HCCs in mice [6,10,11].Natarajan et al. described the use of 89 Zr-labeled humanized αGPC3 for HCC detection in a patient-derived xenograft model [16].We built on this important work by humanizing our radioimmunoconjugate (αGPC3 H ) and performing in vitro and novel in vivo comparisons to its murine predecessor.Here, we report that 89 Zr-αGPC3 H targets GPC3 comparably to 89 Zr-αGPC3 M , resulting in highly speci c tumor uptake and successful HCC detection.

Materials and Methods
Creative Biolabs, Inc. (Shirley, NY) constructed αGPC3 H by engrafting of the parental murine antibody's complementarity-determining region (CDR).Flow cytometry was used to evaluate in vitro binding of αGPC3 M , a chimeric intermediary (αGPC3 C ), αGPC3 H , and αGPC3deferoxamine (DFO) to HepG2 cells.Orthotopic xenograft models of HCC were generated as previously described in athymic nude mice (Jackson Laboratories) [10][11][12]22].Two weeks after HepG2 cell liver injection, bioluminescence imaging (BLI) was used to estimate tumor establishment.αGPC3 was conjugated with DFO and labeled with 89 Zr [10].(For simplicity, 89 Zr-DFO-αGPC3 is written as 89 Zr-αGPC3.)Mice (n = 11 per group) were injected retro-orbitally with 8.1 to 10 megabecquerels (MBq) of 89 Zr-αGPC3 H or 89 Zr-αGPC3 M .Mice with tumors predicted using BLI (n = 6 per group) underwent PET/CT ve days after 89 Zr-αGPC3 injection.Maximum activity concentration (MBq/mL) was measured in a 2D region of interest (ROI) to calculate tumor radioisotope uptake (percent injected dose per milliliter, %ID/mL), tumor-to-liver ratio, and tumor maximum standardized uptake value (SUV max ).Biodistribution studies using gamma counts were performed separately in non-tumor-bearing, non-imaged mice two days after injection and in PET-imaged mice after imaging completion to determine %ID/g for select organs and tumors. Liers from PET-imaged mice were processed for histopathology.Details provided in Supplementary Methods.

Humanized aGPC3 and αGPC3-DFO maintains GPC3 binding in vitro
Binding to HepG2 cell surface GPC3 by unconjugated αGPC3 M , αGPC3 C , and αGPC3 H was con rmed by ow cytometry (Fig. 1a).Binding of DFO-conjugated and αGPC3 M to GPC3 was overall similar to the unconjugated antibody (Fig. 1b).Binding of αGPC3 H and αGPC3 C to GPC3 was greater than αGPC3 M .

Bioluminescence imaging predicts tumor establishment
Tumors were identi ed with BLI (Fig. 2).Mice were assigned to 89 Zr-αGPC3 H and 89 Zr-αGPC3 M injection such that mean photon emission (photons/sec) in tumor-containing ROIs was similar between groups (Table 1).αGPC3 H is amenable to 89 Zr radiolabeling The radiochemical purity of both 89 Zr-αGPC3 antibodies was > 98% and the speci c activity was 0.14 GBq/mg.Details provided in Supplementary Methods.

Discussion
Humanized αGPC3 speci cally targeted GPC3 in vitro and in vivo, enabling HCC detection with immunoPET in an orthotopic xenograft mouse model.This proof-of-concept study builds on our prior research validating a murine radioimmunoconjugate for a theranostic approach to HCC, with potential to improve diagnosis, treatment, and survival [6, [10][11][12].
Our results demonstrate that humanization of 89 Zr-αGPC3 did not alter the highly avid binding to GPC3 on HepG2 cells and liver tumor xenografts.First, ow cytometry established at least equivalent, if not greater, binding of αGPC3 H to GPC3 compared with αGPC3 M , with minimal change when conjugated with DFO.Next, quality assurance of 89 Zr labeling con rmed that 89 Zr-αGPC3 H maintained high purity and speci c activity.The majority of our experiments focused on the novel in vivo comparison between 89 Zr-αGPC3 H and 89 Zr-αGPC3 M .Five of six tumors in each group were detected by immunoPET, with no difference between groups in mean IVIS bioluminescence.PET/CT data revealed no signi cant difference in mean tumor uptake and tumor-to-liver ratios (%ID/mL).Similarly, biodistribution analysis showed no difference in mean organ uptake, tumor uptake, and tumor-to-liver ratios (%ID/g).
While nding comparability between 89 Zr-αGPC3 H and 89 Zr-αGPC3 M achieved the study's primary goal, additional details are worth noting.
First, tumor uptake varied based on tumor size, with higher uptake in larger tumors as previously demonstrated [6].%ID/g (gamma counter) results were greater than %ID/mL (PET) due to limited PET/CT spatial resolution causing partial volume effect; hence, there could be a larger discrepancy between %ID/g and %ID/mL values in mice with smaller tumors (e.g., H3, M9) (Fig. 3, Table 1).While further consideration of the clinical impact of partial volume effect is warranted, this nding does highlight the successful detection of small tumors with 89 Zr-αGPC3 immunoPET.Second, background liver uptake was greater in the 89 Zr-αGPC3 H group, which could imply Fc-mediated liver uptake of 89 Zr-αGPC3 H .However, our prior studies of mice injected with 89 Zr-αGPC3 M compared with non-GPC3-targeting and GPC3-blocked controls demonstrated similar background liver uptake [6,11,16].Furthermore, the tumor-to-liver ratio by nature adjusts for such variables, with no difference between groups suggesting that tumor uptake was also proportionally higher in the humanized antibody group.In fact, tumor-toliver ratios of 12 or greater indicate 89 Zr-αGPC3 H is highly speci c for GPC3-expressing tumors [16].
Tumor presence was histopathologically con rmed in mice with PET-identi ed tumors, while no tumors were found on histologic analysis of livers without PET-identi ed tumors.A limitation here is that, while meticulous gross examination of the liver and histopathologic analysis of suspected tumors was performed, serial sectioning of the entire left hepatic lobe was not undertaken due to limited funding.Therefore, the discordance between BLI and PET for H6 and M12 is unresolved.Of note, the three-week interval between imaging modalities was longer than in previous studies and thus tumor involution may have occurred.
Our study is similar to those from other groups in that it underscores the potential of human αGPC3 to detect HCC with immunoPET, however, there are key differences.Tumor-to-liver ratios by PET/CT and biodistribution analyses were notably higher than those reported by Natarajan et al. using a similar 89 Zr-labeled human αGPC3 IgG antibody and Fayn et al. using 89 Zr-labeled GPC3-targeting HN3 single-domain antibodies.In addition, there was a greater relative difference between tumor uptake and uptake in organs such as the heart, lungs, gastrointestinal tract, and kidneys on biodistribution analysis [16,17].While different methods for model development and radioimmunoconjugate injection used may affect the results such that they are not directly comparable [16,17], it is possible that our humanized antibody has a higher speci city for GPC3-expressing tumors.Furthermore, it should be noted that tumor-to-liver ratios were measured ve days after injection in this study compared with one to seven days after injection in the aforementioned studies, however our prior experiments with 89 Zr-αGPC3 M demonstrated high tumor-to-liver ratios calculated from four hours up to seven days after injection [6, humane use of animals and was carried out in compliance with the ARRIVE guidelines.Speci cally, the minimum number of mice needed to achieve experimental goals were used.Cages were supplied with su cient bedding, food, water, and enrichment throughout the experiment.Appropriate anesthetic/analgesic, ophthalmic lubricant, heating pads, and sterile surgical technique were used for survival surgical procedures to minimize animal distress.Mice were monitored closely during and immediately after surgery for adequate depth of anesthesia and full recovery, respectively; isotonic saline was administered subcutaneously for uid support as needed.Mice were evaluated daily for the rst week after surgery for signs of distress, weight loss, agitation, sedation, lameness, failure to eat/drink, poor or aberrant grooming, abnormal posture, wound, or skin ulcerations.A composite of general appearance, body condition score (BCS), body weight, and abdominal girth measurements were used to assess well-being.After the rst week, mice were evaluated three times weekly for the remainder of the experiment.For bioluminescence imaging and PET/CT, mice were anesthetized and cared for as described in theSupplementary Methods.
Both the research team and Veterinary Services were actively involved in evaluation and care of the mice throughout the experiment.At the completion of the experiment, mice were euthanized using CO 2 followed by cervical dislocation according to IACUC protocol.

Table 1
Mouse-speci c immunoPET and biodistribution data correlated with tumor size a Not measured due to minimal apparent signal.b H6, M12: no tumor identi ed on PET/CT or histopathology (blank = not applicable).c Rank from largest (1) to smallest (10/11).d -426 included in Fig. 4c calculations; -21 excluded from Fig. 4d (see Supplementary Methods).